ABSTRACT This is a proposal to develop a novel high throughput assay system for detection of inhibitors of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA). HBV cccDNA is essential to the virus life cycle, its elimination during chronic infection is considered critical to durable therapy but has not been achieved by the FDA approved small molecule antiviral drugs that exclusively target the viral polymerase. However, because of the limitations of current HBV tissue culture systems, including the impracticality of detecting cccDNA itself, cccDNA has not been rigorously targeted in high throughput screening (HTS) of small molecule libraries. In this proposal, a novel tissue culture line that expresses HA epitope-tagged hepatitis B e antigen (HBeAg) in a cccDNA-dependent manner will be used to develop a cell-based HTS assay for discovery of cccDNA inhibitors. This cell line inducibly produces viral pregenome transcripts from a stably integrated HBV genome (transgene) with an HA epitope sequence inserted in the precore region, leading to replication of viral DNA genome and cccDNA formation; subsequently, HA-tagged-HBeAg RNA and protein are only made from transcripts produced from the cccDNA template, then HA-HBeAg is secreted into the cell culture supernatant. The incorporation of HA-tag into HBeAg is to avoid the cross reaction of HBeAb with HBcAg in the ELISA-based immunological assays, such as chemiluminescence ELISA and AlphaLISA. In an HTS campaign, compounds that lower the HA-HBeAg would be considered candidate inhibitors of cccDNA formation, expression or longevity. Through collaboration with the professional HTS team in the Purdue Chemical Genomics Facility (PCGF), we will first miniaturize the antiviral assay to the 384-well format, the performance characteristics and the robustness of the assay under HTS conditions, including Z?, will be determined. Next, a pilot screen will be conducted against a PCGF sublibrary containing 10,560 ?cherry-picked? compounds, the first round hits will be filtered through dose-ranging activity and cytotoxicity analyses, and through counter-screening in a cell line constitutively expressing transgene- (not cccDNA-) dependent HA-HBeAg to remove the off-target hits. Finally, the HTS-derived hits will be evaluated in cccDNA-producing cell lines by directly measuring the levels of cccDNA and/or its transcripts. The confirmed hits and their analogs will also be resynthesized and retested for their activity against cccDNA to obtain the final hits. Thus, the accomplishment of this project will deliver an HTS platform for discovery of novel HBV inhibitors from larger compound libraries; the pilot screen and hit validation effort will expand the pool of compounds to be used for HBV research, or even the possible derivation of transformational therapies for chronic hepatitis B.